Calcium-dependent protease (calpain) exists in most mammalian cell types. This enzyme has two major isoforms that differ in their sensitivity to calcium ions (calpain I and calpain II) (see Murachi T., Biochem Int 1989;18:263-294). Calpain resides in the cytosol of cells and is activated by Ca.sup.2+ at physiological pH. Its proteolytic activity appears to be selective against certain target proteins, such as components of the cytoskeleton and calmodulin-dependent enzymes. To date, there are no high affinity, selective, nonpeptide inhibitors of calpain reported (see Wang K. K. W., Trends Pharmacol Sci 1990;11:139-142).
Excessive excitation by a neurotransmitter glutamate can lead to death of nerve cells (neurons) and neurodegeneration (see Meldrum B., Garthwaite J., Trends Pharmacol Sci, Special Report 1991, 1991:54-62. It is believed that toxic effect of glutamate comes from overactivation of its target glutamate receptors (e.g., under ischemic conditions or stroke). This in turn produces an influx of calcium ion (Ca.sup.2+) into the neurons. The rise of cellular Ca.sup.2+ level triggers the activation of calpain. Calpain then goes on to degrade cytoskeletal protein such as spectrin, which is believed to disrupt normal cellular functions, and eventually leads to cell death (Siman R., Noszek J. C., Kegerise C., J Neurosciences 1989;9:1579-1590). Inhibition of calpain by products covered by the present invention minimizes the cellular damage and therefore prevents neurodegeneration. Several nonselective calpain inhibitors were shown to be neuroprotective in various ischemia models (Arai A., Kessler M., Lee K. S., Lynch G., Brain Res 1990;532:63-68).
Abnormal protein processing is implicated in Alzheimer's disease and calpain and several of its target proteins including the amyloid precursor protein and tau protein have been identified as major components of Alzheimer's neurofibrillary tangles (Holtzman D. M., Mobley W. C., Trends Biochem Sci 1991;16:140-144; Iwamoto N., Thangnipon W., Crawford C., Emson P. C., Brain Res 1991:177-180) in affected regions of the brain. Amyloid precursor protein has also been found to be sensitive to calpain digestion (Siman R., et al, J Neuroscience 1990;10:2400-2411). It is conceivable that calpain activity may be defective that leads to abnormal processing of its target protein. It argues that inhibition of calpain by products covered in the present invention will provide therapeutical benefits to the patients of Alzheimer's disease.
Cataract is an opacity occurring in the lens as a result of a variety of insults to the lens (Shearer T. R., David L. L., Anderson R. S., Current Eye Res 1987;6:289-300). In an experimental model where cataract is induced by overdose of sodium selenite (selenite cataract), it has been shown that the lens shows increase of calcium and water-insoluble proteins. Calpain is also found in high concentration in corneal lens epithelium. In selenite cataract, it was demonstrated that lens proteins such as .alpha.-, .beta.-crystallins, and cytoskeletal proteins were degraded during cataract formation (Marcantonio J. M., Duncan G., Biochem Soc Trans 1991;19:1148-1150). Cysteine protease inhibitor E64, which inhibits calpain, has been shown to reduce the rate of cataract formation in whole animal (Azuma M., David L. L., Shearer T. R., Current Eye Res 1991;10:657-666). Calpain inhibitors such as the compounds covered in the present invention can be of use in retarding cataract formation.
It has been demonstrated that intracellular calcium level rises after traumatic injury to brain or spinal cord (Banik N. L., Hogan E. L., Whetstine L. J., Balentine J. D., 1984;1:131-137). Overactivation of calpain is supposed to play a role on the degenerative process that occurs after the injury, similar to the excitatory amino acid toxicity in the brain (Taft W. C., Lyeth B. G., Dixon C. E., Hayes R. L., Soc Neurosciences 1991:164, Abstr. 17). There is also evidence of calpain degradation of myelin protein which can cause neurodegeneration (Banik N., McAlhaney W. W., Hogan E. L., J Neurochemistry 1985;45:581-588). Calpain inhibitors of the present invention can minimize the degeneration observed.
Ischemic myocardiac infraction is a result of blockade of blood supply in the coronary vessels. It has been reported that calpain activation was observed in the cardiac myocytes during these conditions (Iizuka K., Kawaguchi H., Yasuda H., Biochemical Medicine and Metabolic Biology 1991;46:427-431) coronary reperfusion of cysteine protease inhibitor E64c after acute myocardiac infraction in dogs was found to significantly reduce the size of infract (Toda G., Matsushita S., Kuramoto K., et al, Jap Heart J 1989;30:375-386). Again, calpain inhibitors in the present invention can provide therapeutic benefits.
Percutaneous transluminal coronary angioplasty is now a widely accepted medical procedure to expand the inner diameter of clotted artery in atherosclerotic coronary artery disease patients. However, the success rate of this procedure is dampened by the spontaneous, slow renarrowing of the arteries (restenosis) (McBride W., Lange R. A., Hillis L. D., New Eng J Med 1988;318:1734-1737). Recently, it has been shown that controlling proliferation and migration of smooth muscle cells to the neoinitimal layer of the blood vessel by calpain inhibitor I and other cysteine protease inhibitors (March K. L., Roeske R., Hathaway D. R., Clin Res 1990;38:234A). It was further shown that these agents can inhibit restenosis (Wilensky R. L., March K. L., Hathaway D. R., J Amer Coll Cardiol 1991;17:268A). Since the compounds in this invention are also calpain inhibitors, they should be useful in reducing angioplastic restenosis.
Calpain is found in the synovial (joint) fluid of the knee joint. In fact, both calpain I and II in the synovial fluid were found to increase by several-fold in rheumatoid arthritis patients (Fukui I., Tanaka K., Murachi T., Biochem Biophys Res Commun 1989;162: 559-566). A major cartilage component proteoglycan was also found to be a calpain substrate (Suzuki K., et al, Biochem J 1992;285:857-862). It is believed that calpain overactivation has a damaging effect of the joint and in the inflammation process itself. Inhibition of calpain activity by compound in the present instant invention may provide therapeutic benefit to the patients suffering from such inflammation.
Disruption in the regulation of intracellular calcium concentration was reported in muscular dystrophy, such as Duchenne muscular dystrophy (DMD) and after muscle denervation. Intracellular calcium levels in muscular dystrophic mice was found to be significantly raised which resulted in increased protein degradation. Many myofibrillar proteins (such as myosin, Troponin I and T) are indeed good calpain substrates (Sugita H., Ishiura S., Kamakura K., Nakase H., Hagiwara K., Nonaka I., In: Calcium Regulation in Biological Systems, (Ebashi S., Endo M., Imahori K., Kakiuchi S., Nishizuka Y., eds, Academic Press, 1984:243-257.) and calpain II concentration was higher in dystrophic skeletal muscle in animals (Johnson P., Hammer J. L., Int J Biochem 1988;20:1227-30). Therefore, it is conceivable that calpain overactivation plays an important role in abnormal myofiber degradation. Calpain inhibitors described in this invention will be useful treatment in muscular degenerative disorders such as muscular dystrophy or muscle denervation.
Formation of blood clots (thrombosis) is a result of platelet aggregation. When platelets are stimulated by thrombin or plasmin, two key events occur which lead to platelet aggregation (Puri R. N., et al, Am J Physiol 1990;259:C862): 1) putative ADP-receptor aggregin is proteolyzed and 2) fibrinogen receptors become exposed on the platelet surface. Aggregin was hydrolyzed in vitro by calpain but not by thrombin. It was further found that thrombin increased intracellular calcium levels in platelets thus activating calpain which then hydrolyzed aggregin. Calpain also appeared to modify platelet membrane structure thus exposing latent fibrinogen receptor (Baldassare J. J., et al, J Biol Chem 1985;260:10531-10535). This allows fibrinogen binding to platelet that leads to aggregation. It was shown that calpain inhibitors Phe-Gln-Val-Val-Cys(3-nitro-2-thiopyridine)-Gly-NH.sub.2 and high molecular weight kininogen blocked aggregin breakdown and platelet aggregation (Puri R. N., et al, Am J Physiol 1990;259:C862; Puri R. N., et al, Blood 1991;77:500-507). Therefore, calpain inhibitors described in this patent should be effective platelet aggregation inhibitors.